RESUMEN
Protein quality control is accomplished by inducing chaperones and proteases in response to an altered cellular folding state. In Escherichia coli, expression of chaperones and proteases is positively regulated by sigma32. Chaperone-mediated negative feedback control of sigma32 activity allows this transcription factor to sense the cellular folding state. We identified point mutations in sigma32 altered in feedback control. Surprisingly, such mutants are resistant to inhibition by both the DnaK/J and GroEL/S chaperones in vivo and also show dramatically increased stability. Further characterization of the most defective mutant revealed that it has almost normal binding to chaperones and RNA polymerase and is competent for chaperone-mediated inactivation in vitro. We suggest that the mutants identify a regulatory step downstream of chaperone binding that is required for both inactivation and degradation of sigma32.
Asunto(s)
Escherichia coli/genética , Proteínas de Choque Térmico/genética , Factor sigma/genética , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiología , Retroalimentación , Regulación Bacteriana de la Expresión Génica , Chaperonas Moleculares/genética , Chaperonas Moleculares/fisiología , Mutación , Plásmidos , Mutación PuntualRESUMEN
We compare the elongation behavior of native Escherichia coli RNA polymerase holoenzyme assembled in vivo, holoenzyme reconstituted from sigma70 and RNA polymerase in vitro, and holoenzyme with a specific alteration in the interface between sigma70 and RNA polymerase. Elongating RNA polymerase from each holoenzyme has distinguishable properties, some of which cannot be explained by differential retention or rebinding of sigma70 during elongation, or by differential presence of elongation factors. We suggest that interactions between RNA polymerase and sigma70 may influence the ensemble of conformational states adopted by RNA polymerase during initiation. These states, in turn, may affect the conformational states adopted by the elongating enzyme, thereby physically and functionally imprinting RNA polymerase.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , Factor sigma/química , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/fisiología , Conformación Proteica , Factor sigma/fisiologíaRESUMEN
The heat shock response controls levels of chaperones and proteases to ensure a proper cellular environment for protein folding. In Escherichia coli, this response is mediated by the bacterial-specific transcription factor, sigma32. The DnaK chaperone machine regulates both the amount and activity of sigma32, thereby coupling sigma32 function to the cellular protein folding state. In this manuscript, we analyze the ability of other major chaperones in E. coli to regulate sigma32, and we demonstrate that the GroEL/S chaperonin is an additional regulator of sigma32. We show that increasing the level of GroEL/S leads to a decrease in sigma32 activity in vivo and this effect can be eliminated by co-overexpression of a GroEL/S-specific substrate. We also show that depletion of GroEL/S in vivo leads to up-regulation of sigma32 by increasing the level of sigma32. In addition, we show that changing the levels of GroEL/S during stress conditions leads to measurable changes in the heat shock response. Using purified proteins, we show that that GroEL binds to sigma32 and decreases sigma32-dependent transcription in vitro, suggesting that this regulation is direct. We discuss why using a chaperone network to regulate sigma32 results in a more sensitive and accurate detection of the protein folding environment.
Asunto(s)
Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/fisiología , Respuesta al Choque Térmico/fisiología , Chaperonas Moleculares/fisiología , Pliegue de Proteína , Factor sigma/fisiología , Transcripción Genética , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
FtsH, a member of the AAA family of proteins, is the only membrane ATP-dependent protease universally conserved in prokaryotes, and the only essential ATP-dependent protease in Escherichia coli. We investigated the mechanism of degradation by FtsH. Other well-studied ATP-dependent proteases use ATP to unfold their substrates. In contrast, both in vitro and in vivo studies indicate that degradation by FtsH occurs efficiently only when the substrate is a protein of low intrinsic thermodynamic stability. Because FtsH lacks robust unfoldase activity, it is able to use the protein folding state of substrates as a criterion for degradation. This feature may be key to its role in the cell and account for its ubiquitous distribution among prokaryotic organisms.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de la Membrana/química , Factor sigma , Proteasas ATP-Dependientes , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Proteínas Fluorescentes Verdes , Proteínas de Choque Térmico/metabolismo , Cinética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Plásmidos/metabolismo , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , ARN Bacteriano/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleasas/metabolismo , Especificidad por Sustrato , Temperatura , Termodinámica , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
All cells have stress response pathways that maintain homeostasis in each cellular compartment. In the Gram-negative bacterium Escherichia coli, the sigma(E) pathway responds to protein misfolding in the envelope. The stress signal is transduced across the inner membrane to the cytoplasm via the inner membrane protein RseA, the anti-sigma factor that inhibits the transcriptional activity of sigma(E). Stress-induced activation of the pathway requires the regulated proteolysis of RseA. In this report we show that RseA is degraded by sequential proteolytic events controlled by the inner membrane-anchored protease DegS and the membrane-embedded metalloprotease YaeL, an ortholog of mammalian Site-2 protease (S2P). This is consistent with the mechanism of activation of ATF6, the mammalian unfolded protein response transcription factor by Site-1 protease and S2P. Thus, mammalian and bacterial cells employ a conserved proteolytic mechanism to activate membrane-associated transcription factors that initiate intercompartmental cellular stress responses.